The present invention relates to a process of compatibly administrating carcinostatic substances and compatibly administrative carcinostatic substances, and especially to a process of compatibly administrating carcinostatic substances in which the synergistic effect appears by compatible administration, compatibly administrative carcinostatic substances employable in the practice of the above process and a mixed carcinostatic substance prepared by mixing these carcinostatic substances. The present invention further relates to a process of rapidly inspecting the carcinostatic substances.
A carcinostatic substance is generally administrated compatibly with another carcinostatic substance for elevating its effectiveness and for preventing tumor cells from obtaining a resistance against a medicine. However, no special method has existed for suitably selecting the administrative substance. The administrative substance has been selected depending on the experience of a clinician under the general considerations on {circle around (1)} an administrative substance is also effective when employed by itself and {circle around (2)} a side effect is not overlapped with that of the administrative substance.
A reaction for restoring from the damage induced by a medicine (carcinostatic substance) is derived in the tumor cell, and if the recovering mechanism is not sufficiently realized, the tumor cell becomes extinct. However, a certain period of time is required for achieving the extinction (necessity of additional administration). When two or more kinds of carcinostatic substances are compatibly employed, a cell biological reaction (synergistic effect) may take place which cannot be recognized in single administration, and in this case the distinct actions of the respective carcinostatic substances (not completely elucidated) may competitively appear. Various administrations conducted under the different conditions such as a dosage of the compatible agents and a dosage time are necessary for elucidating the above mechanism.
The present inventor has developed a reasonable technique in connection with the compatible administration of carcinostatic substances (CANCER CHEMOTHERAPY; Challenge for the Future, vol. 4, 1989; Molecular Biology of DNA Topoisomerases. Proceedings of the International Symposium on DNA Topoisomerases in Chemotherapy, 1991), and proved the effect of the compatible administration of the various carcinostatic substances. However, the combination of the carcinostatic substance which produces the synergistic effect is scarce so that the satisfactorily compatible effect of the carcinostatic substances cannot be easily obtained.
Medicines are roughly divided in accordance with an administration means into two groups one of which is directed to the administration through an intravenous injection and the other is directed to the pororal administration. Most of conventional platinum carcinostatic substances are perorally administrated so that a large burden is applied to a patient.
In the meantime, a cell exists from its birth to its death performing various morphological changes especially a nucleic change. It is interesting to monitor the morphological changes in view of molecular biology. In order to conduct this monitoring, a microscope is generally employed so that the condition of the cell is visually observed. However, in this commonly employed microscope observation, the morphological changes inside of the cell cannot be monitored so that the employment of fluorescence dye has been proposed.
However, the fluorescence dye destroys the cell after it is bonded to the cell so that the continuous observation of the morphological changes of the living cell can be impossible even though the morphosis of the cell at the time of employing the fluorescence dye may be observed.
In order to overcome this drawback, the present inventor had investigated various kinds of the fluorescence dye so as to propose 4xe2x80x2,6-diamidino-2-phenylindolexe2x80xa22HCl (hereinafter referred to as xe2x80x9cDAPIxe2x80x9d) as fluorescence dye which may be employed for continuously observing the morphological changes of living cells without extinguishing the cells even if it is bonded to the cell [Article in General Meeting of Japan Cancer Institute (July 1990) page 390, 1946).
On the other hand, cell division is an especially interesting phenomenon among the morphological changes, and the the process of the cell division is classified and divided into a division prophase, a division metaphase, a division anaphase and a division telophase. FIGS. 1 to 9 show the conditions of the cell division from the division prophase at the time of initiation of the cell division to the cell division telophase in turn. The left sides of the respective figures are microphotographs of 1500 magnifications of the cell division taken without employing fluorescence dye. Among these, FIGS. 1 to 3 show the cells of the division prophase, FIGS. 4 to 6 show the cells of the division metaphase. FIG. 7 shows the cells of the division anaphase and FIGS. 8 and 9 show the cells of the division telophase.
The cells of FIGS. 1 to 9 are under the conditions of the cell division, but only the cells of FIGS. 6 to 9 can be recognized to be in the cell division through the ordinary microscope observation employing specimens fixed by means of a fixative. The cells of FIGS. 1 to 6 cannot be recognized only through the ordinary microscope observation.
The easy recognition whether the cell is in the condition of the cell division or not is important not only for the scientific purpose but also for the confirmation purpose of the effectiveness of the various medicines.
However, as mentioned, among the several phases of the cell division, the division conditions of the cells in the division prophase and in the period from the first half to the beginning of the second half of the division metaphase cannot be recognized through the ordinary microscope observation so that the detailed study thereof is impossible.
Frequent occurrence of the various organ cancers invites vital developments of anti-tumor substances or carcinostatic substances, and in these developments, it is essential to rapidly conduct the inspection of the effectiveness of the carcinostatic substances (sensibility test) for reducing the development expenses.
The above inspection test is roughly divided into two methods. A first method essentially consists of cultivating tumor cells, cultivating the tumor cells in a test tube with carcinostatic substances and judging the degree of the extinction after the extinction of the cells, and a second method essentially consists of injecting or perorally administrating carcinostatic substances to an animal transplanted with tumor cells and comparing the numbers of survival days of the animals for judging the effectiveness of the carcinostatic substances.
The judgment of the effectiveness of the carcinostatic substances cannot be performed before the extinction of the cells of the death of the animals in each of the above methods and between one and four weeks are ordinarily required for the judgment so that not only the rapid judgment is impossible but also the expenses for continuing the test increase.
Although the present inventor has proposed a method of more rapidly inspecting the effectiveness of medicines (Japanese patent laid open gazette No. 58-184547), not less than four days and between seven days and four weeks are required in the test tube method and in the animal method, respectively, for the judgment.
Further in these methods, only the effectiveness of medicines or the extinction effect is made clear and how the carcinostatic substance functions to extinct the cancer cells (mechanism) cannot be specified.
Although the above method employing DAPI as fluorescence dye has enabled the visual observation of the process of cell extinction by means of the carcinostatic substance while keeping the cells alive, the effectiveness of the carcinostatic substance cannot be confirmed without the observation of the cell extinction in the said process so that between three and six days are required.
In view of the above drawbacks, an object of the present invention is to provide a process of compatibly administrating cis-oxalato (1R,2R-diaminocyclohexane) Pt (II) (hereinafter referred to as xe2x80x9cl-OHPxe2x80x9d) which is a new carcinostatic substance with one or more existing carcinostatic substances.
Another object of the present invention is to provide the above process in which the optimum combination of the carcinostatic substances is proposed.
A further object of the present invention is to provide a process of compatibly administrating a new platinum complex with one or more existing carcinostatic substances for further elevating the anticancer property of a carcinostatic substance a main component of which is the above platinum complex.
A still further object of the present invention is to provide a process of easily distinguishing cells not only in the division anaphase and the division telophase in the cell division but also in the division prophase and the division metaphase and of observing their morphosis.
A still further object of the present invention is to provide a process of inspecting the effectiveness of carcinostatic substances utilizing the above process of distinguishing the cells.
A first aspect of the present invention is a process of compatibly administrating a carcinostatic substance which comprises compatibly administrating one or more compatible agents selected from a group consisting of cisplatin, carboplatin, 5-fluorouracil (hereinafter referred to as xe2x80x9c5-FUxe2x80x9d), tegaful, carmoful, doxifluridine, uracil, irinotecane, adriamycin, etoposide, mitomycin, mitoxisantrone and bleomycin with l-OHP.
A second aspect of the present invention is a carcinostatic substance selected from a group consisting of cisplatin, carboplatin, 5-FU, tegaful, carmoful, doxifluridine, uracil, irinotecane, adriamycin, eloposide, mitomycin, mitoxantrone and bleomycin which can be compatibly administrated with l-OHP. This carcinostatic substance may prepared by simply mixing the cisplatin or the like and the l-OHP or by making a coordination bond between them.
A third aspect of the present invention is a process of compatibly administrating a carcinostatic substance which comprises compatibly administrating one or more compatible agents selected from a group consisting of cisplatin, vincristin, carboplatin, 5-FU, tegaful, carmoful, doxifluridine, uracil, irinotecane, adriamycin, etoposide, mitomycin, mitoxantrone and bleomycin with a platinum complex having a Formula I [in this Formula, a Formula II is a diamine selected from a group consisting of 1,2-cycloalkane (C5 to C7) diamine {its stereostructure is cis-(R,S-), trans-d(1S,2S-) or trans-l (1R,2R-)} having a Formula III, 2-aminomethlcyclohexylamine {its stereostructure is cis-l (R,R-), cis-d(S,S-), trans-l (R,S-) or trans-d(S,R-)} having a Formula IV and 1,1-diaminomethylcyclohexane, 0-phenylenediamine, ethylenediamine or propyrenediamine having a Formula V, and a Formula VI is a ligand forming a five or six-membered ring coordinated to Pt(IV) in 0xe2x80x940 coordination, and X is an aliphatic alkyl or an aromatic alkyl having C1 to C10]. 
A fourth aspect of the present invention is a carcinostatic substance comprising the compatible agent and the platinum complex of the third aspect.
A fifth aspect of the present invention is a process of observing cell division which comprises adding fluorescence dye to cells to be observed, bonding the fluorescence dye to each cell, and observing the cells with a microscope of clarifying the cell division condition.
A sixth aspect of the present invention is a process of rapidly inspecting a carcinostatic substance which comprises adding fluorescence dye and a carcinostatic substance to be inspected to a plurality of cancer cells which have been cultivated, continuing the cultivation, measuring the number of the cells in the division period appearing in the plurality of the cancer cells and inspecting the effect of the carcinostatic substance.
In accordance with the first aspect of the present invention, a shown in the below Examples, the compatible administrations of the l-OHP and the existing carcinostatic substances to the tumor cells exhibit. In almost all the cases, an additive effect of extinguishing the tumor cells and, in the most cases, exhibit a synergistic effect.
The increase of the extinguishing effect of the tumor cells obtained by the compatible administration of the carcinostatic substances is significantly meaningful especially in the range competing with the multiplication rate of the tumor cells, and even a slight increase of the extinguishing effect may largely promote the therapeutic effect.
The increase of the extinguishing effect in accordance with the above process of compatibly administrating the carcinostatic substances is made to be epoch-making by suitably selecting the combination of the carcinostatic substances so that this process can considerably contribute to the most cancer therapies.
The l-OHP and the compatible agent may be simultaneously administrated or either of them may be added later than the other, and the timing of the administration may be determined depending on the combination.
However, in order to reduce the burden of a patient and a physician, the simultaneous administration is desirable, and the 5-FU of which an effect produced by its simultaneous administration is twice an expected value is an especially effective compatible agent.
The compatibly administrative carcinostatic substance in accordance with the second aspect possess the remarkable therapy effect against the cancer as mentioned in connection with the first aspect.
In case of the combination of the carcinostatic substances preferably administrated simultaneously, desirably a pellet may be formed or an injection ampoule may be prepared with the mixture of the l-OHP and one or more compatible agents because the labor of the compatible administration may be omitted.
In accordance with the third aspect of the present invention similar to the first aspect, the compatible administrations of the platinum complex having a Formula VII and the existing carcinostatic substances to the tumor cells exhibit, in almost all the cases, an additive effect of extinguishing the tumor cells and, in the most cases, exhibit a synergistic effect.
The compatibly administrative carcinostatic substance in accordance with the fourth aspect possesses the remarkable therapy effect against the cancer as mentioned in connection with the third aspect. 
The fifth aspect of the present invention enables the visual observation of the divided cells under the division prophase and the division metaphase which are difficult to be visually observed by adding the fluorescence dye which can be bonded to ordinary cells and cancer cells under the cultivation and bonding the dye to the said cells, and in other words, enables the observation not only for of the shape of the divided cell but also of the change of a chromosome so that the observation of the said divided cell under the division prophase and the division metaphase in which an external change may seldom occur becomes possible.
In accordance with this observation method, the number of the cells under the cell division among a plenty of cells can be accurately counted so that a percentage of the cells under the cell division to all the cells can be calculated.
If the carcinostatic substance to be inspected exhibits the same number of the cells under the cell division as that of a control having no carcinostatic substance, the said carcinostatic substance to be inspected possesses no anti-tumor property. If, on the other hand, the said substance exhibits the smaller or larger number than that of the control, the carcinostatic substance to be inspected possesses the anti-tumor property so that the effectiveness as the carcinostatic substance can be judged by counting the number of the cells under the cell division and comparing the number with that of the control.